Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a major health problem responsible for 1.7 million deaths each year. The emergence of multiply and extensively drug resistant strains of Mtb makes the situation even more alarming as it raises concern about untreatable tuberculosis. There is an urgent need to develop new drugs to treat tuberculosis. Many bacterial pathogens possess a twin arginine translocation (Tat) pathway that exports proteins to the bacterial cell envelope or host environment. Mammalian cells do not have a Tat pathway, making this pathway an attractive candidate for novel antimicrobials. The Tat pathway of Mtb is a particularly compelling drug target: it is an essential pathway in Mtb, it exports Mtb virulence factors, and it exports the Mtb ?-lactamase that imparts resistance to ?-lactam antibiotics. In this project we will develop robust high-throughput screening (HTS) assays for identifying inhibitors of the Mtb Tat pathway. Whole-cell phenotypic assays will be developed using the fast-growing and nonpathogenic model mycobacteria M. smegmatis (Msmeg). The Tat pathway of Msmeg is not essential but it is necessary for ?-lactam resistance. In Aim 1, we will develop a primary screening assay for Tat inhibitors that is based on the ?-lactam sensitivity of Msmeg mutants lacking the Tat pathway. In Aim 2, we will develop secondary assays to distinguish compounds that elicit ?-lactam sensitivity through Tat inhibition as opposed to other mechanisms. Assays for establishing target-specificity and for testing on virulent Mtb will also be developed. In Aim 3, w will perform pilot screening to assess the effectiveness of our assays and the screening cascade with hits progressing to testing on Mtb. The compounds identified through this research will help to validate the Mtb Tat pathway as a novel drug target and they will also be valuable as the first chemical probes for studying the Tat pathway of bacteria. Upon completion of this work, we intend to apply for Fast Track entry of our screening campaign into the NIH Molecular Libraries Probe Production Centers Network.